Multiphoton laser scanning microscopy advantages and disadvantages
Multi-Photon Laser Scanning Microscopy is an experimental method based on laser scanning microscopy technology that provides more accurate optical sectioning capabilities in 3D viewing. The multiphoton fluorescence excitation method uses long wavelengths of red or near-infrared light to acquire high-resolution fluorescent images of specimens with minimal killing of active specimens, making it suitable for imaging living cells, especially thick living tissues such as brain slices, embryos, whole organs and even whole organisms.
Advantages are as follows:
1, the use of red light or infrared light excitation, light scattering is small (the scattering of small particles and the wavelength of the fourth power of the inverse ratio).
2, does not require a pinhole, can collect more scattered photons from the imaging cross-section.
3, the pinhole can not distinguish between the scattered photons emitted by the out-of-focus region or focal area, multi-photon in the deep imaging signal-to-noise ratio is good.
4, single-photon excitation of ultraviolet or visible light used in the beam to reach the focal plane before the sample is easily absorbed and attenuated, not easy to deep excitation.
5, in the biological microscope observation, * the first consideration is not to damage the active state of the organism itself, to maintain water, ion concentration, oxygen and nutrient circulation. In light observation occasions, both thermal and photon energy must stay in the cell without damage to the amount of irradiation, light energy.
6, multi-photon microscope also has many advantages. Such as three-dimensional resolution, depth intrusion, in the scattering efficiency, background light, signal-to-noise ratio, control, etc., there are previous laser microscopes do not have or have incomparable beyond the characteristics.
Multi-photon confocal laser scanning microscope has been extended to various research and application fields. It is capable of three-dimensional non-destructive observation of samples in their natural state, and can improve the resolution and signal-to-noise ratio of the system. Using the change of material properties after multi-photon excitation, it is also possible to achieve three-dimensional height data storage and three-dimensional microfabrication in any direction, which has a high application value. It can be believed that with the further development of machinery, materials, laser technology and other technologies related to multiphoton confocal microscopy, multiphoton confocal laser scanning microscope will be greater development and wider application.
Disadvantages are as follows:
1, only fluorescence imaging.
2, if the sample includes chromophores that can absorb the excitation light, such as pigments, the sample may be thermally damaged.
3, the resolution is slightly reduced, although it can be improved by the simultaneous use of confocal aperture, but there will be signal loss.
4. The cost of multiphoton scanning microscopy is high due to the limitation of expensive ultrafast lasers.
